Abstract The goal of this proposal is to elucidate the precise functions of two major subtypes of somatostatin- expressing inhibitory interneurons (SST INs) in controlling branch-specific dendritic Ca2+ spikes and synaptic plasticity of layer 5 (L5) pyramidal neurons during motor skill learning. Our preliminary studies show that different motor learning tasks induce dendritic Ca2+ spikes on different apical tuft branches of L5 pyramidal neurons in the mouse primary motor cortex. Furthermore, we found that inactivation of SST INs disrupts branch-specific generation of Ca2+ spikes. We have also found that different subtypes of SST INs in deep cortical layers target different domains of the apical dendrite of L5 pyramidal neurons and have specific patterns of in vivo activity during spontaneous brain state transitions. With a combination of experimental approaches including channelrhodopsin-assisted patching (ChAP), three dimensional two-photon imaging, intersectional and pharmacogenetic manipulations of the activities of specific subtypes of SST INs, we propose to test the hypothesis that SST IN subtypes with specific axonal targeting regulate branch-specific dendritic Ca2+ spikes in the motor cortex that are critical for the induction and maintenance of synaptic plasticity in motor learning. In Aim 1 we will use ChAP to efficiently record from SST INs in deep cortical layers and investigate the patterns of activity of distinct SST IN subtypes during the motor skill learning behavior. These experiments will provide for the first time the pattern of activity of SST INs in deep cortical layers in motor cortex during motor skill learning. Since the ChAP method also allows the targeted labeling of specific cell types for post hoc morphological analysis, the experiments will permit us to correlate the patterns of in vivo activity of SST cells with their axonal targeting properties. In Aim 2 we will use pharmacogenetics in intersectional mouse lines that target SST INs with specific innervation patterns to manipulate the activity of two major subtypes of SST INs that exert differential inhibition onto the apical tuft branches of L5 pyramidal neurons. These experiments will allow us to investigate the mechanisms underlying dendritic branch-specific Ca2+ spike generation and synaptic plasticity. The proposed experiments will reveal the fundamental role of SST INs in branch-specific dendritic Ca2+ spikes and in the induction and maintenance of synaptic plasticity during learning and memory formation. These studies may provide novel insights into how dysfunction of inhibitory circuits causes intellectual disability and autism.